With inherent optical sectioning capability, deeper penetration and reduced out-of-focus photodamage, multiphoton microscopy technologies, especially two-photon fluorescence (TPF) and second harmonic generation (SHG), have been widely used in biological and biomedical studies. However, their benefits to in vivo studies and clinical applications are limited, since the conventional bench-top laser-scanning microscope (LSM) lacks access to tissues deep inside animal bodies. To extend the applicability of these high-resolution powerful imaging modalities, a miniature flexible endomicroscope with imaging capability comparable to standard LSMs is highly desirable, and recent years have witnessed a plethora of different endomicroscope designs.
Irrespective of the variety of optical and mechanical designs, one central challenge to build an endomicroscope for practical clinical usage is to achieve as high system detection sensitivity as possible. Here the detection sensitivity is quantitatively defined as the achievable signal-to-noise ratio (SNR) per unit incident power per unit fluorophore concentration, which is basically an inclusive metric of the system's capability to acquire high-SNR two-photon images. For clinical applications, high detection sensitivity is particularly critical as: 1) it is preferable to utilize just endogenous fluorophores, e.g. nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), and/or structural proteins, e.g. collagen fibers, all generally existing in lower abundance and exhibiting much smaller two-photon cross sections compared with exogenous dyes or fluorescent proteins; 2) the maximal applicable incident power is limited due to safety concerns. Previous endomicroscopy prototypes, although demonstrating great potential, generally suffered from inadequate sensitivity, as either sample staining or very high excitation power (˜70 mW) was required for adequate image quality.
The difficulty to achieve high detection sensitivity is partially aggravated by the miniaturization. Several previous designs employed two optical fibers: one single-mode fiber for excitation light delivery and one large-diameter multimode fiber for signal collection. Such dual-fiber scheme, although favorable for collecting more nonlinear emission, usually led to a probe size too large to go through the access channels (˜2.8-4 mm diameter) of commercial gastroscopes or colonoscopes.
It would therefore be advantageous to provide a smaller probe that provides high quality images and still fits within the access channels of commercial scopes.